To understand the molecular mechanism(s) by which the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) promotes growth and differentiation, the structure and activity of two cellular thyroid hormone binding proteins have been studied. I. Role of phosphorylation on the transcriptional activity of human beta1 thyroid hormone nuclear receptors (h-TRbeta1). h-TRbeta1 was found to be a phosphoprotein in vivo with Ser, Thr and Tyr (85:10:5) as the phosphorylation sites. Okadaic acid, a potent inhibitor of phosphatase 1 and 2A, stimulated the phosphorylation of h-TBbeta1 by 3-, 7- and 11-fold at the concentrations of 0.1, 0.25 and 0.5 muM, respectively. The increase in phosphorylation was accompanied by a concomitant increase in receptor- mediated transcription in transient transfection assays. h-TRbeta1 purified from E. coli was phosphorylated in vitro by the endogenous kinase from cellular extracts. Ser, Thr and Tyr were phosphorylated in a similar ratio to that found in vivo. The in vitro phosphorylation was stimulated by okadaic acid. Phosphorylation did not affect the binding of h-TRbeta1 to T3. However, phosphorylation of h-TRbeta1 resulted in an increase of its binding to DNA and conferred on it the ability to bind to nuclear accessory proteins. Taken together, these results indicate that phosphorylation plays an important role in the transcriptional activity of h-TRbeta1. II. The cytosolic thyroid hormone binding protein (p58-M2) regulates the h-TRbeta1-mediated transcription. The transcriptional activity of h- TRbeta1 is T3-dependent. To explore the possible role of p58-M2 as a regulator for T3 action, we evaluated the effect of the availability of cytoplasmic T3 on the modulation of transcriptional responses of T3 receptor. In human choriocarcinoma JEG-3 and monkey COS-1 cells, p58-M2 is a monomer of the tetrameric pyruvate kinase, subtype M2(PKM2) which does not bind T3. The in vivo monomer-tetramer interconversion is regulated by glucose via fructose 1,6-bisphosphate. At the physiological T3 concentration, lowering the glucose concentration led to an increase in the cellular concentration of p58-M2 and a concomitant reduction in the transcriptional activity of a transfected h-TRbeta1 in both cell lines. In the absence of glucose, the transcriptional activity of h-TRbeta1 in JEG-3 and COS-1 cells was reduced by 65-75% and 90-95%, respectively. These findings demonstrate an important prenuclear step in the modulation of the gene regulating activity of the T3 receptors.